Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

Abstract

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca²⁺) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Ca_v blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca²⁺]_cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca²⁺ pools, and Ca²⁺ influx by ORAI channels have been associated with the increase in [Ca²⁺]_cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca²⁺]_cyt. To address these questions, we monitored changes in [Ca²⁺]_cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca²⁺]_cyt at concentrations above 10 μM (IC₅₀ around 50 μM) and a fast Ca²⁺ increase capacity at 100 μM. We found that inhibiting IP₃ receptors, depleting the ER-Ca²⁺ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca²⁺]_cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP₃R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Keywords: ALC cells; Ca2+ signaling; Cav; HEK293 cells; LS8 cells; Mibefradil; PLC pathway.

Fig. 1

Effects of mibefradil on [Ca²⁺]_cyt transients in LS8, ALC, and HEK-293 cells. a [Ca²⁺]_cyt transients in LS8 cells stimulated with mibefradil (0.1 μM to 100 μM) in the presence of 2 mM extracellular Ca²⁺. b LogEC₅₀ (apparent affinity) plot. c Quantification of ∆Ca²⁺ peak. d-f Same as in a-c but measured in ALC cells. g-i Same as a-c but measured in HEK-293 cells. In all cells, traces show basal (0–120 s) Ca²⁺ levels and the maximum capacity of Ca²⁺ mobilization (240–300 s) at 100 μM. Data represent the mean ± SEM of ≥75 cells from 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *P < 0.05 vs. MIB-100 μM group; ^@P < 0.05 vs. MIB-50 μM group; ^#P < 0.05 vs. MIB-10 μM group; n.s., non-significant

Fig. 2

IP₃ content and expression of IP₃ receptor subtypes. a Quantification of IP₃ content in LS8 cells. b Quantification of IP₃ content in ALC cells. c Quantification of IP₃ content in HEK-293 cells. In all cells, quantification was performed before and after incubation with mibefradil (100 μM, 3–4 min). d-f Relative mRNA expression of the ITPR gene encoding the IP₃R subtypes 1–3 quantitated by qPCR in each cell type. Data represent the mean ± SEM of 3–4 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *P < 0.05; **P < 0.01 or ***P < 0.001 vs. IP₃R-1 group; ^@P < 0.05 vs. IP₃R-2 group; n.s., non-significant

Fig. 3

Effects of mibefradil are reduced or abolished by IP₃ R antagonist and ER-Ca²⁺ depletion. a Original traces of [Ca²⁺]_cyt transients in Xestospongin C treated LS8 cells in Ca²⁺-free Ringer’s solution before and after mibefradil (100 μM) stimulation. b Quantification of the ∆ Ca²⁺ peak. c Quantification of the Ca²⁺ influx rate. d-f Same as in a-c but in ALC cells. g-i Same as in a-c but in HEK-293 cells. j Original traces of [Ca²⁺]_cyt transients in CPA stimulated LS8, ALC, and HEK-293 cells in Ca²⁺-free Ringer’s solution followed by the application of mibefradil (100 μM). k Quantification of the ∆ Ca²⁺ peak under CPA stimulation. i Quantification of the ∆ Ca²⁺ peak under mibefradil stimulation. Data represent the mean ± SEM of ≥52 cells from 3 independent experiments. Data were analyzed by two-tailed unpaired Student’s t-test. *P < 0.05 or **P < 0.01 vs. respective MIB-100 μM (control) group; n.s., non-significant. In ER-Ca²⁺ depletion experiments elicited by CPA (20 μM), the data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. ^&P < 0.05 vs. LS8 group n.s., non-significant

Fig. 4

Mibefradil-mediated [Ca²⁺]_cyt increase is dependent on PLC pathway activation. a Original traces of [Ca²⁺]_cyt transients in LS8, ALC, and HEK-293 cells stimulated with the PLC activator m-3M3FBS (60 μM), followed by addition of mibefradil (100 μM) in Ringer’s solution containing 2 mM Ca²⁺. b Quantification of the ∆Ca²⁺ peak area of under m-3M3FBS stimulation. c Quantification of the ∆Ca²⁺ peak under mibefradil stimulation. d Original traces of [Ca²⁺]_cyt transients in LS8 and ALC cells stimulated with mibefradil (100 μM) in the presence or absence of the PLC inhibitor, U73122 (5 μM). d Quantification of the ∆Ca²⁺ peak. f Quantification of the Ca²⁺ influx rate. g-i Same as d-f but in HEK-293 cells. Data represent the mean ± SEM of ≥101 cells from 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. ^#P < 0.05 vs. LS8 group; *P < 0.05 vs. their respective MIB-100 μM group; n.s., non-significant

Fig. 5

Mibefradil stimulated ER-Ca²⁺ depletion and Ca²⁺ influx via SOCE. a Original traces of [Ca²⁺]_cyt transients showing ER-Ca²⁺ depletion elicited by mibefradil (100 μM) in Ca²⁺-free Ringer’s solution followed by readdition of 2 mM extracellular Ca²⁺ solution in LS8 cells with or without pre-incubation with synta-66. b Quantification of the ER-Ca²⁺ release peak area under mibefradil stimulation. c Quantification of ∆SOCE peak upon readdition of 2 mM Ca²⁺. d Same as in a-c but in ALC cells. g Original traces of [Ca²⁺]_cyt transients showing ER-Ca²⁺ depletion elicited by mibefradil (100 μM) in Ca²⁺-free Ringer’s solution followed by readdition of 2 mM extracellular Ca²⁺ solution in HEK-293 cells with a CRISPR-cas9 deletion of ORAI1 (SKO) or dual deletion of ORAI2 and ORAI2 (DKO). h Quantification of the ER-Ca²⁺ release peak area under mibefradil stimulation. i Quantification of ∆SOCE peak upon readdition of 2 mM Ca²⁺. Data represent the mean ± SEM of ≥93 cells from 3 independent experiments. Data were analyzed by two-tailed unpaired Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *P < 0.05 vs. ALC group; **P < 0.01 vs. HEK-control group; ***P < 0.001 vs. LS8 group; ^#P < 0.05 vs. HEK-ORAI1 group; n.s., non-significant